1. Field of the Invention
This invention relates to an endothelial cell harvesting kit which allows one to rapidly obtain viable human endothelial cells. Previous research efforts have been directed to develop antithrombogenic linings in synthetic vascular prostheses by endothelial cell seeding. In order to develop these linings, natural endothelial cells must first be extracted from segments of human arteries and veins.
2. Background of the Invention
Endothelial cell harvesting is a process for removing viable endothelial cells from a portion of autologous vein. Cell harvesting is accomplished by mechanical scraping or rubbing, or enzymatic treatment. One use for the harvested cells is to "seed" vascular prostheses.
Extensive animal experimentation and limited experience in humans indicate that seeded grafts endothelialize more quickly, induce less platelet reaction, and have better patency than nonseeded grafts. Adjunctive antiplatelet therapy may be required until the endothelial lining has developed. Antiplatelet therapy, however, is a standard procedure for patients receiving vascular grafts.
Briefly, harvesting is performed as follows. A segment of autologous vein is excised from a subject and the endothelial cells are removed. The cells so derived are concentrated by centrifugation, suspended in an aliquot of the subject's blood, and placed in the vascular graft until a thin layer of clot is formed on the graft wall. The "seeded" graft is immediately implanted in the subject. Endothelial cells "seeded" into the graft subsequently grow to confluence on the luminal surface thus providing the graft with a true neointima. Only autologous cells are used in this process.
Researchers at several universities have investigated methods of harvesting human cells for seeding. At the University of Indiana, a method was developed which involved the mechanical harvesting of canine endothelial cells. In those studies, endothelial cells were obtained by passing a steel wool pledget through excised vein segments. Mechanical harvesting methods have a derivation efficiency of less than about 75% and endothelial cells obtained in this manner are often contaminated with smooth muscle cells. (Herring, M. B. et al., "A Single-staged Technique For Seeding Vascular Grafts With Autogenous Endothelium." Surgery, 84:498-504 (1978)).
Another mechanical method for harvesting endothelial cells requires isolating a part of the aorta and placing it in sterile Hanks' Balanced Salt Solution (HBSS). The vessels were then further chemically treated, slit open and the endothelial cells were removed by gentle scraping with a scalpel blade. The collected cells were centrifuged and resuspended and further treated to suppress growth of contaminating smooth muscle cells (Sharefkin, J. et al., "Endothelial Cell Labeling With Indium-III-Oxine As A Marker of Cell Attachment To Bioprosthetic Surfaces." Journal of Biomedical Materials Research, 17:345-357 (1983)).
Another technique for harvesting human cells is by enzymatic means. Researchers at the University of Michigan used this method to obtain canine endothelial cells for seeding purposes. Here, cells were derived from segments of external jugular veins. According to this technique, freshly excised veins were secured and everted over a stainless steel rod and suspended in iced HBSS. The veins were then washed of any adherent blood cells by spinning with a centrifuge in fresh HBSS. After washing, the veins were incubated in a solution of tryspin, HBSS and ethylenediaminetetracetic acid (EDTA) and then a solution of collagenase, HBSS, calcium and magnesium. The vein with culture medium, enzyme, and wash solution were spun and centrifuged to obtain endothelial cells. (Graham, L. et al., "Expanded PTFE Vascular Prostheses Seeded With Enzymatically Derived And Cultured Canine Endothelial Cells," Surgery, 91:550-559 (1982)). Such enzymatic harvesting methods have a derivation efficiency between about 80 to nearly 100%. However, cells isolated by this method are often injured due to the required eversion of the vein.
Researchers at Harvard University have developed a technique where human saphenous veins are cannulated and then filled with collagenase and submerged in HBSS, The veins were then flushed several more times with collagenase. Dislodged cells were colleced by centrifugation. (Watkins et al., "Adult Human Saphenous Vein Endothelial Cells: Assessment Of Their Reproductive Capacity For Use In Endothelial Seeding Of Vascular Prosthesis, " Journal of Surgical Research, 36:588-596 (1984)).
Personnel at the University of Pennsylvania have also isolated endothelial cells from an umbilical vein by using a technique which included using a tapered glass cannula to which the vein was fastened. Initially, the vein was filled with collagenase and allowed to incubate. After incubation, one syringe was filled with medium 199 (M199) supplemented with antibiotics (gentamicin and amphotericin B). The syringes were pushed back and forth so that the vessel contents were similarly moved. The slight mechanical agitation promoted the release of endothelial cells that were subsequently centrifuged, resuspended and further treated for seeding. (Macarak, E. et al., "Adhesion of Endothelial Cells To Extracellular Matrix Proteins," Journal Of Cellular Physiology, 116:76-86 (1983)).
At Cornell University, techniques have also been developed to obtain endothelial cells from umbilical veins. That method required that the vein be cannulated at one end and flushed with HBSS. The other end was then cannulated and the vein was flushed with more HBSS. The vein was then filled with collagenase to distend it and incubated. After incubation, the cord was gently kneaded between the fingers up and down its length to increase cell yield. The collagenase solution was flushed out of the vein and centrifuged. The cells formed a pellet and they were resuspended for seeding. (Jaffe E., "Biology of Endothelial Cells," 1984, Martinus Nijhoff Publishers).
Researchers at Universitant Giessen have also investigated the use of Dispase I for isolating endothelial cells from the human umbilical cord vein. Here, human umbilical cords were flushed with Dulbecco-phosphate-buffered saline (D-PBS) to remove excess blood and then filled with Dispase I and incubated for 15 minutes. The vein was then flushed with D-PBS, centrifuged at room temperatures and the pellet was then resuspended for harvesting. (Thilo, D.G.S., et al., "Isolation Of Human Venous Endothelial Cells By Different Proteases," Artery, 8(3):259-266, 1980).
To date no reports averse to the seeding process or to the use of endothelial-cell-seeded grafts have appeared in the literature. In addition, no articles pertaining to the use of a kit for harvesting cells have been published.